The long-term objective of this proposal is to understand how intracellular signals are directed along specific pathways to induce a defined output. Signal transduction pathways typically function in interconnected web-like networks composed of common or "shared" signaling modules. An important question is how does a signal follow a particular path through a common module to induce a specific response? Some diseases like cancer can arise because a signal that meant to follow one path is misdirected to another. The proposal focuses on the Rho GTPase Cdc42 in the model eukaryote, Saccharomyces cerevisiae. In S. cerevisiae, Cdc42 is a major regulator of multiple signaling pathways, but it is not clear how Cdc42 induces a specific response in one setting and a completely different response in another. Importantly, Cdc42 also functions in determining the axis of cell polarity, and this provides a clue to its specificity. A recent discovery from our lab showed that proteins required for establishing polarity also direct Cdc42 function to a specific MAPK pathway. This discovery is important because it links positional information with the cell's decision-making process. This proposal will attempt to uncover the molecular mechanisms for how positional information is used to direct pathway specificity. Aim1 will test how recruitment of the Cdc42 activating protein (GEF) by polar landmarks controls MAPK activation. This will be done by using a combination of genetic and cell biological approaches. Aim 2 will use molecular and biochemical approaches to investigate the role of a Cdc42-interacting protein (Bem4) as a pathway-specific scaffold for the MAPK pathway. The potential mechanism(s) by which Bem4 regulates Cdc42 will be tested. Aim3 focuses on a newly- identified cell-surface protein (Opy2) that acts with a signaling mucin to stimulate Cdc42. Mucins are important but poorly understood proteins whose function is mechanistically distinct from the well-studied GPCR class of MAPK regulators. How the Opy2 mucin adaptor activates Cdc42 will be determined. PUBLIC HEALTH RELEVANCE: Signal transduction pathways regulate a wide variety of cellular responses and when mis-regulated cause diseases such as cancer. We have discovered new regulators of the polarity regulatory GTPase Cdc42 that regulates a cell differentiation pathway. Understanding how Cdc42 is activated in a particular context to restructure cell polarity in a precise manner will help generally to understand GTPase regulation and specification.